Sperm-specific cation channel, catsper4, and uses therefor

ABSTRACT

Nucleic acid and protein sequences relating to a cation channel which is sperm-specific (CatSper4) are disclosed. The CatSper4 protein is shown to be specifically expressed in sperm. Nucleic acids, vectors, transformed cells, transgenic animals, polypeptides, and antibodies relating to the CatSper4 gene and protein are disclosed. Also provided are methods of in vitro fertilization and contraception, methods of identifying modulators of CatSper4 activity, methods of genotyping subjects with respect to CatSper4, and methods of diagnosing and treating CatSper4-mediated disorders, including infertility. Related business methods are also disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/458,643, filed Aug. 13, 2014, which is a continuation of U.S.application Ser. No. 13/371,033, filed Feb. 10, 2012, which is acontinuation of U.S. application Ser. No. 12/244,630, filed Oct. 2,2008, which is a divisional application of U.S. application Ser. No.10/523,475, filed Sep. 16, 2005, which claims benefit of priority to PCTInternational Application No. PCT/US2003/024359, filed Aug. 4, 2003 andU.S. Provisional Application Ser. No. 60/402,115, filed Aug. 7, 2002,the contents of which are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the fields of molecular biology, andreproductive technology. In particular, the invention relates to acation channel protein expressed specifically in sperm cells, to nucleicacids encoding the protein, cells engineered to express the protein,assays for compounds affecting the activity of the protein, and to theuse of such compounds in the treatment or causation of infertility, oras a means of contraception or animal control.

Description of the Related Art

Sperm and ova reciprocally interact in mammalian fertilization(Wassarman, et al. (2001), Nature Cell Biology 3, E59-E64; Yanagimachi(1994), in The Physiology of Reproduction, eds. Knobil & Neill (RavenPress, New York), pp. 189-315). To reach the site of fertilization,sperm must travel relatively long distances and become primed forfertilization of the ova through capacitation and other processes. Oncethey arrive at the surface of an ovum, sperm interact with ovumextracellular matrix glycoproteins including the zona pellucidaproteins. Sperm release acidic material during the acrosome reaction, asignaling event that presumably involves the opening of Ca²⁺ channelsand the influx of Ca²⁺ into the sperm heads (O'Toole, et al. (2000), MolBiol Cell 11, 1571-84). The TRPC2 protein, a putative Ca²⁺-permeantchannel, has recently been implicated in the acrosome reaction(Jungnickel, et al. (2001), Nat Cell Biol 3, 499-502). Penetration ofsperm through the thick outer layer of the ovum is achieved throughchemical lysis of the ovum coat and/or the mechanical motion of sperm(Bedford (1998), Biol Reprod 59, 1275-87). Following infiltration of theovum ZP coat, the sperm membrane fuses with that of ovum. Fusion isfollowed by activation of the fertilization process, beginning with Ca²⁺oscillations in the ovum (Wassarman, et al. (2001), Nature Cell Biology3, E59-E64; Yanagimachi (1994), in The Physiology of Reproduction, eds.Knobil & Neill (Raven Press, New York), pp. 189-315).

Ca²⁺ and cyclic nucleotides control sperm motility (Tash (1990) inControls of Sperm Motility: biological and clinical aspects, ed. Gagnon(CRC Press, Boca Raton), pp. 229-240; Darszon, et al. (1999), PhysiolRev 79, 481-510; Hyne & Garbers (1979), Proc Natl Acad Sci USA 76,5699-703) and several voltage-dependent Ca²⁺ channel (Ca_(V)) mRNAs andcyclic nucleotide-gated (CNG) channel proteins have been detected insperm cell precursors (Darszon, et al. (1999), Physiol Rev 79, 481-510;Serrano, et al. (1999), FEBS Lett 462, 171-6; Weyand, et al. (1994)Nature 368, 859-63; Wiesner, et al. (1998), J Cell Biol 142, 473-84).Furthermore, low voltage activated, dihydropyridine-sensitive “T-type”channels (Santi, et al. (1996), Am J Physiol 271, C1583-93; Arnoult, etal. (1996), Proc Natl Acad Sci USA 93, 13004-9) and pharmacologicallydefined N- and R-type currents have been measured in spermatogenic cells(Wennemuth, et al. (2000), J Biol Chem 275, 21210-7). But the role ofthese channels in spermatogenesis or mature sperm function is not known.

Two sperm-specific cation channels, designated CatSper1 and CatSper2,have previously been described (Ren et al. (2001), Nature 413:603-9;Quill et al. (2001), Proc. Natl. Acad. Sci. (USA) 98(22):12527-31).These proteins are the subject of U.S. Prov. Appln. Ser. No. 60/288,402,filed May 3, 2001, U.S. Prov. Appln. Ser. No. 60/327,167, filed Oct. 4,2001, U.S. Prov. Appln. Ser. No. 60/345,324, filed Oct. 22, 2001, andPCT Intl. Appln. No. PCT/US02/13487, filed May 3, 2002,

SUMMARY OF THE INVENTION

In one aspect, the present invention provides isolated nucleic acidscorresponding to all or a portion of a CatSper4 gene. In someembodiments, the isolated nucleic acids include a nucleotide sequence ofat least 10, 12, 14, 16 or 18 consecutive nucleotides of SEQ ID NO: 1 orSEQ ID NO: 3, or a sequence complementary thereto. In other embodiments,the nucleic acids include nucleotide sequences encoding a CatSper4protein, at least a transmembrane domain of a CatSper4 protein, at leastan extracellular loop of a CatSper4 protein, at least a pore region of aCatSper4 protein, at least an epitope of a CatSper4 protein having highpredicted antigenicity, or a sequence complementary thereto. Inparticular embodiments, the nucleic acids include a sequence of SEQ IDNO: 1; a sequence of SEQ ID NO: 3; a sequence encoding a polypeptidecomprising residues 46-75, 86-98, 108-137, 155-202, 209-233 and 242-272of SEQ ID NO: 2; a sequence encoding a polypeptide comprising residues48-73, 81-111, 113-134, 156-204, 208-233 and 245-273 of SEQ ID NO: 4; asequence encoding a polypeptide comprising residues 76-85, 138-154 and234-242 of SEQ ID NO: 2; a sequence encoding a polypeptide comprisingresidues 74-80, 135-155 and 234-244 of SEQ ID NO 4; a sequence encodinga polypeptide comprising residues 222-231 of SEQ ID NO: 2; a sequenceencoding a polypeptide comprising residues approximately residues222-231 of SEQ ID NO: 4; a sequence encoding a polypeptide comprising ahigh predicted antigenicity epitope of SEQ ID NO: 2; a sequence encodinga polypeptide comprising a high predicted antigenicity epitope of SEQ IDNO: 4; and a sequence complementary thereto.

In another aspect, the invention provides isolated nucleic acidsencoding polypeptide having at least 80%, 85%, 90%, or 95% amino acidsequence identity with a CatSper4 protein; at least a transmembranedomain of a CatSper4 protein; at least an extracellular loop of aCatSper4 protein; and at least a pore region of a CatSper4 protein. Insome embodiments, the isolated nucleic acids encode a polypeptide havingat least 80%, 85%, 90% or 95% amino acid sequence identity with aCatSper4 protein and having CatSper4 activity in a cell capable ofexpressing CatSper4 activity. In some embodiments, the isolated nucleicacids include a regulatory element having at least 80%, 85%, 90% or 95%nucleotide sequence identity to at least 100, 200, 300 or 400consecutive nucleotides from SEQ ID NO: 5, and that is capable ofpromoting transcription of a coding sequence operably joined thereto ina mammalian cell in which a CatSper4 gene can be expressed.

In another aspect, the invention provides isolated nucleic acids thathybridize to at least a portion of a nucleic acid of SEQ ID NO: 1 or SEQID NO: 3 under conditions including a wash step of 1.0×SSC at 65° C., awash step of 0.5×SSC, a wash step of 0.2×SSC, or a wash step of 0.1×SSC.In some embodiments, the isolated nucleic acids encode a polypeptidehaving CatSper4 activity.

In another aspect, the invention provides nucleic acid comprising anucleotide sequence encoding a polypeptide having CatSper4 activity, andthat hybridizes to at least a portion of a nucleic acid of SEQ ID NO: 1or SEQ ID NO: 3 under conditions including a wash step of 1.0×SSC at 65°C., a wash step of 0.5×SSC, a wash step of 0.2×SSC, or a wash step of0.1×SSC; and that is operably joined to a heterologous regulatory regionsuch that the sequence is expressed. In another embodiment, theinvention provides a nucleic acid comprising a nucleotide sequenceencoding a polypeptide having at least 80%, 85%, 90% or 95% amino acidsequence identity with an amino acid sequence of SEQ ID NO: 2 or 4; andis operably joined to a heterologous regulatory region such that thesequence is expressed.

In another aspect, the invention provides a kit for detecting at least aportion of a CatSper4 nucleic acid. The kits can include any of theforegoing isolated nucleic acids of the invention, and a means fordetecting the isolated nucleic acid. In some embodiments, the means fordetecting the isolated nucleic acid includes a detectable label boundthereto and, in some embodiments, the means includes a labeled secondarynucleic acid which specifically hybridizes to the first isolated nucleicacid.

In another aspect, the invention provides vectors including any of theforegoing isolated nucleic acids of the invention. In some embodiments,the vector includes a genetic construct capable of expressing thenucleic acids of the invention. In some embodiments, the nucleic acidsof the invention are operably joined to an exogenous regulatory regionand, in some embodiments, the nucleic acids are operably joined toheterologous coding sequences to form a fusion vector. In someembodiments, the vector includes a CatSper4 regulatory region and, insome embodiments, the CatSper4 regulatory region is operably joined to aheterologous coding sequence.

In another aspect, the invention provides cells transformed with theforegoing nucleic acids of the invention, or a genetic construct capableof expressing a nucleic acid of the invention. In some embodiments, thenucleic acid of the invention is operably joined to heterologous codingsequences to encode a fusion protein. In some embodiments, the cells arebacterial cells, yeast cells, insect cells, nematode cells, amphibiancells, rodent cells, or human cells. In some embodiments, the cells aremammalian somatic cells, fetal cells, embryonic stem cells, zygotes,gametes, germ line cells and transgenic animal cells.

In another aspect, the invention provides non-human transgenic animals.In these aspects, a genetic construct has introduced a modification intoa genome of the animal, or an ancestor of the animal, and themodification includes insertion of a nucleic acid encoding at least afragment of a CatSper4 protein, inactivation of an endogenous CatSper4gene, or insertion by homologous recombination of a reporter geneoperably joined to CatSper4 regulatory elements. In some embodiments,the modification is insertion of nucleic acid encoding a CatSper4protein, at least a transmembrane domain of a CatSper4 protein, at leastan extracellular loop of a CatSper4 protein, at least a pore region of aCatSper4 protein, or at least an epitope of a CatSper4 protein havinghigh predicted antigenicity. In some embodiments, the animals are rats,mice, hamsters, guinea pigs, rabbit, dogs, cats, goats, sheep, pigs, andnon-human primates.

In another aspect, the invention provides substantially pure proteinpreparations including polypeptides selected from a CatSper4 protein; atleast a transmembrane domain of a CatSper4 protein; at least anextracellular loop of a CatSper4 protein; at least a pore region of aCatSper4 protein; and at least an epitope of a CatSper4 protein havinghigh predicted antigenicity. In particular embodiments, the polypeptideis selected from SEQ ID NO: 2; SEQ ID NO: 4; residues 46-75, 86-98,108-137, 155-202, 209-233 and 242-272 of SEQ ID NO: 2; residues 48-73,81-111, 113-134, 156-204, 208-233 and 245-273 of SEQ ID NO: 4; residues76-85, 138-154 and 234-242 of SEQ ID NO: 2; residues 74-80, 135-155 and234-244 of SEQ ID NO 4; residues 222-231 of SEQ ID NO: 2; residues222-231 of SEQ ID NO: 4; a high predicted antigenicity epitope of SEQ IDNO: 2; and a high predicted antigenicity epitope of SEQ ID NO: 4.

In another aspect, the invention provides a substantially pure proteinpreparation including polypeptides having at least 80%, 85%, 90%, or 95%amino acid sequence identity with a CatSper4 protein; at least atransmembrane domain of a CatSper4 protein; at least an extracellularloop of a CatSper4 protein; or at least a pore region of a CatSper4protein. In some embodiments, the substantially pure protein preparationincludes a polypeptide having at least 80%, 85%, 90%, or 95% amino acidsequence identity with a CatSper4 protein and having CatSper4 activityin a cell capable of expressing CatSper4 activity.

In another aspect, the invention provides a substantially pure antibodypreparation including an antibody raised against a CatSper4 epitope. Insome embodiments, the epitope has high predicted antigenicity. In someembodiments, the epitope includes an amino acid sequence within a highpredicted antigenicity epitope of SEQ ID NO: 2, and a high predictedantigenicity epitope of SEQ ID NO: 4. In some embodiments, the antibodyis a monoclonal antibody. In some embodiments, antibody is an Fabfragment, an F(ab′)₂ fragment, an Fv fragment, or a single-chain Fvfragment (scFv).

In another aspect, the invention provides a kit for detecting at leastan epitope of a CatSper4 protein. The kits include an anti-CatSper4antibody of the invention and a means for detecting said antibody. Insome embodiments, the means for detecting said anti-CatSper4 antibodyincludes a detectable label bound thereto and, in some embodiments, themeans for detecting said anti-CatSper4 antibody includes a labeledsecondary antibody which specifically binds to the anti-CatSper4antibody.

In another aspect, the invention provides methods of identifyingpotential modulators of CatSper4 activity. The methods includecontacting a candidate compound with a cell expressing a CatSper4protein; measuring an indicator of CatSper4 activity in the cell;determining whether the candidate compound caused an increase ordecrease in the indicator relative to a reference level; and identifyingthe candidate compound as a potential modulator of CatSper4 activity ifthe increase or decrease is significant. In some embodiments, theindicator is an indicator of the level of mRNA encoding the CatSper4protein, an indicator of the level of CatSper4 protein, an indicator ofcation flux across a membrane of said cell, or an indicator of wholecell or channel currents of said cell. In some embodiments, the cell hasbeen transformed with a genetic construct capable of expressing aCatSper4 protein. In some embodiments, the cell is a mature sperm celland the indicator is a measure of sperm motility.

In another aspect, the invention provides methods of identifying apotential modulator of CatSper4 activity comprising contacting acandidate compound with at least a structural domain of a CatSper4protein; measuring binding, if any, between the candidate compound andthe CatSper4 moiety; and identifying the candidate compound as apotential modulator of CatSper4 activity if the binding is significant.In some embodiments, the CatSper4 moiety is a CatSper4 protein; at leasta transmembrane domain of a CatSper4 protein; at least an extracellularloop of a CatSper4 protein; or at least a pore region of a CatSper4protein.

In another aspect, the invention provides a method of decreasing thefertility of a male subject by administering a compound to the subjectwhich decreases CatSper4 activity. In another aspect, the inventionprovides a method of causing reversible infertility in a male subject byadministering to a compound to the subject which decreases CatSper4activity. In another aspect, the invention provides a method ofcontraception in which a compound which decreases CatSper4 activity isadministered to a male or female subject. In each of the foregoingembodiments, the compound can be in an injection, a transdermal patch, abioerodable implant, a lubricant, a moisturizer, a foam, a jelly, or asponge. If the subject is a female, the compound can be administeredinto at least one of the vagina, uterus or fallopian tubes. In each ofthe foregoing embodiments, the compound can be a nucleic acid which isantisense to at least a portion of a CatSper4 gene or an antibody to aCatSper4 protein, including an Fab fragment, an F(ab′)₂ fragment, an Fvfragment, or an scFv fragment. In some embodiments, the subject is amammal. In some embodiments, the subjects are humans, dogs, cats, cows,sheep, horses, mice, rats, raccoons, and gophers. In others embodiments,the subjects are fish, amphibians or insects. In related aspects, theinvention provides for the use of a compound which decreases CatSper4activity in the preparation of a medicament for decreasing the fertilityof a male subject, or causing reversible infertility in a male subject,or in the preparation of a contraceptive for administration to a male orfemale. thus, the invention provides contraceptive preparationsincluding compounds which decrease CatSper4 activity, including nucleicacids which are antisense to at least a portion of a CatSper4 gene andantibodies to a CatSper4 protein.

In another aspect, the invention provides method of diagnosing aCatSper4-related disorder in a mammal by determining the presence orabsence of a mutation in a CatSper4 gene. In some embodiments, thepresence or absence of differences between a determined nucleic acid oramino acid sequence and a reference sequence indicates the presence orabsence of mutations in the CatSper4 gene. In some embodiments, themethod includes contacting at least a fragment of the CatSper4 proteinwith an antibody known to bind to a CatSper4 protein in which a mutationis known to be present or absent and detecting binding between theantibody and the CatSper4 protein. In other embodiments, the methodincludes measuring an indicator of CatSper4 activity in a cell; andcomparing the measured indicator to a reference level. The indicator canbe an indicator of the level of mRNA encoding CatSper4 protein, anindicator of the level of CatSper4 protein, an indicator of cation fluxacross a membrane of said cell, or an indicator of whole cell or channelcurrents of said cell. In some embodiments, the disorder isCatSper4-related infertility. In another aspect, the invention providesmethods of genotyping a subject with respect to a CatSper4 gene.

In another aspect, the invention provides a method of in vitrofertilization by sperm having decreased CatSper4 activity, decreasedmotility, or decreased ability to penetrate a zona pellucida, in which azona pellucida is removed from at least one ovum; and the ovum iscontacted with at least one of sperm.

In another aspect, a method of treating a subject characterized byinfertility due to decreased CatSper4 activity is provided. The methodincludes transforming sperm or sperm progenitors of the subject with agenetic construct capable of expressing a CatSper4 protein and usingtransformed sperm of said subject to fertilize an ovum. Alternatively,the method includes administering a CatSper4 protein to sperm or spermprogenitors of the subject.

In another aspect, the invention provides methods of diagnosing ananti-CatSper4 antibody-mediated infertility caused by anti-CatSper4antibodies present in a female urogenital tract is provided. In anotheraspect, methods of treating an anti-CatSper4 antibody-mediatedinfertility caused by anti-CatSper4 antibodies present in a femaleurogenital tract are provided.

In another aspect, the invention provides methods of conducting a drugdiscovery business including (a) identifying, by an assay of theinvention, one or more agents which antagonize CatSper4 activity; (b)determining if an agent identified in step (a), or an analog thereof,inhibits at least one of sperm motility or egg penetrance; (c)conducting therapeutic profiling of an agent identified as an inhibitorin step (b) for efficacy and toxicity in one or more animal models; and(d) formulating a pharmaceutical preparation including one or moreagents identified in step (c) as having an acceptable therapeuticprofile. In some embodiments, the method further includes the step ofestablishing a system for distributing the pharmaceutical preparationfor sale, and optionally including establishing a sales group formarketing the pharmaceutical preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention.

FIG. 1 shows the primary structure of mouse CatSper4 protein. (a) Aminoacid sequence; the 6 putative transmembrane domains are underlined andthe pore region is in bold.

FIG. 2 shows the primary structure of human CatSper4 protein. (a) Aminoacid sequence; the 6 putative transmembrane domains are underlined andthe pore region is in bold.

FIG. 3 shows cladograms of the (a) murine and (b) human CatSper family.

DETAILED DESCRIPTION

The present invention depends, in part, upon the identification,isolation and characterization of a cation channel protein which isexpressed in sperm cells, but not in other tissues tested, and whichplays a significant role in the motility of sperm and their ability tofertilize ova. The protein has been designated CatSper4 to indicate thatit is the fourth Cation channel which is Sperm-specific to beidentified. Other members of the CatSper family of proteins includeCatSper1 (Ren et al. (2001), Nature 413:603-9; U.S. Prov. Appln. Ser.No. 60/288,402, filed May 3, 2001; U.S. Prov. Appln. Ser. No.60/327,167, filed Oct. 4, 2001; PCT Intl. Appln. No. PCT/US02/13487,filed May 3, 2002), CatSper2 (Quill et al. (2001), Proc. Natl. Acad.Sci. (USA) 98(22):12527-31; U.S. Prov. Appln. Ser. No. 60/345,324, filedOct. 22, 2001), and CatSper3 (co-pending U.S. Prov. Appln. Atty. DocketNo. CMC-003PR, filed of even date herewith). Significantly, inhibitionof the activity of CatSper1 has been shown to cause a substantialdecrease in the motility of sperm cells, particularly the most vigoroussperm tail beating required for penetration of the zona pellucida (ZP)and subsequent fertilization, and CatSper1 knock-out mice have beenshown to be infertile. Like CatSper1 and the other members of theCatSper family of proteins, the CatSper4 protein includes sixtransmembrane domains and a pore region, its expression is strictlylocalized to testis (e.g., spermatocytes and the principal piece ofsperm), and its function appears related to cyclic nucleotide-mediatedcalcium entry and motility. Data indicate that the CatSper proteins mayheteromultimerize to function in vivo.

The CatSper4 gene encodes six transmembrane domains and a pore region.The gene product is exclusively expressed in the testis and not in othertissues such as the heart, brain, spleen, lung, liver, skeletal muscle,or kidney. In sperm, the channel is localized primarily to the tail'sprincipal piece, not the head or midpiece.

Agonists and antagonists of the activity of the CatSper4 protein canmodulate sperm motility and, therefore, can be used to treat infertilityor as male and female contraceptives.

The patent, scientific and medical publications referred to hereinestablish knowledge that was available to those of ordinary skill in theart at the time the invention was made. The entire disclosures of theissued U.S. patents, published and pending patent applications, andother references cited herein are hereby incorporated by reference. Inparticular, the entire disclosures of U.S. Prov. Appln. Ser. No.60/288,402, filed May 3, 2001, U.S. Prov. Appln. Ser. No. 60/327,167,filed Oct. 4, 2001, U.S. Prov. Appln. Ser. No. 60/345,324, filed Oct.22, 2001, and PCT Intl. Appln. No. PCT/US02/13487, filed May 3, 2002,are incorporated herein by reference.

DEFINITIONS

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art; references to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques which would be apparent to one of skill in theart. In order to more clearly and concisely describe the subject matterwhich is the invention, the following definitions are provided forcertain terms which are used in the specification.

As used herein, the term “CatSper4 protein” means a sperm-specificcation channel such as the human CatSper4 protein disclosed in SEQ IDNO: 2, human allelic variants of the disclosed CatSper4 protein,mammalian homologs of these human CatSper4 proteins, and functionalequivalents thereof. The term CatSper4 protein refers to naturallyoccurring proteins as isolated from sperm, recombinantly producedproteins from cells transformed with CatSper4 genes, and fusion proteinsin which CatSper4 sequences are fused to N-terminal or C-terminalpolypeptides. The term “fragment” refers to fragments of the CatSper4proteins, such as structural domains and epitopes. A fragment of aCatSper4 protein comprises at least six amino acid residues.

As used herein, the term “CatSper4 gene” means a gene encoding aCatSper4 protein, including the human CatSper4 protein disclosed in SEQID NO: 2, human allelic variants of the disclosed CatSper4 protein,mammalian homologs of these human CatSper4 proteins, and functionalequivalents thereof. The term CatSper4 gene refers to both naturallyoccurring genes as isolated from genomic DNA, and recombinantly producedgenes in which the CatSper4 coding regions are operably joined to eitherendogenous or exogenous regulatory elements, with or without intronsequences, and with or without 5′ or 3′-flanking sequences which canencode heterologous (i.e., non-CatSper4) sequences to form a CatSper4fusion protein. A CatSper4 gene will include, at a minimum, a codingregion encoding the protein operably joined to regulatory elements(e.g., promoters, enhancer) which allow transcription of the codingregion to mRNA which can be translated into a CatSper4 protein.

As used herein “CatSper4” activity means any normal biological activityof a wild-type CatSper4 protein when expressed in a cell or cell type inwhich CatSper4 is normally expressed and under conditions under whichCatSper4 is normally expressed. Such activity can include induction ofan ion current; mediation of cAMP-induced Ca²⁺ influx; restoration ofsperm motility when expressed in CatSper4−/− sperm; and/or restorationof the ability to penetrate eggs when expressed in CatSper4−/− sperm.CatSper4 activity can be measured in sperm cells or spermatocytes, or inother cells in which any necessary accessory factors are present.

As used herein with respect to nucleic acid and amino acid sequences,the term “identity” means a measure of the degree of similarity of twosequences based upon an alignment of the sequences which maximizesidentity and which is a function of the number of identical nucleotidesor residues, the number of total nucleotides or residues, and thepresence and length of gaps in the sequence alignment. A variety ofalgorithms and computer programs are available for determining sequenceidentity using standard parameters. For example, Gapped BLAST orPSI-BLAST (Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402),BLAST (Altschul et al. (1990) J Mol. Biol. 215:403-410), andSmith-Waterman (Smith et al. (1981), J Mol. Biol. 147:195-197). As usedherein, percent identity is based upon the default values for the BLASTalgorithms.

As used herein, the term “homolog” means a protein which isevolutionarily-related to and shares substantial, conserved structuraland functional similarity with a reference protein, but which is presentin a different species (e.g., human, rat, and insect CatSper4 proteinsare homologs of each other).

As used herein, the term “mutation” refers to a change in a nucleic acidsequence, whether or not expressed as a change in a correspondingencoded protein sequence, relative to some reference sequence. Thereference sequence can be a “wild-type” sequence (i.e., one or more highfrequency sequences in a population corresponding to a “normal”phenotype), or any other sequence. As used herein, the term mutation isintended to be synonymous with the term polymorphism, and therefore thedifferences between any two non-identical sequences can be regarded asmutations. The term mutation is intended to encompass insertions,deletions and/or substitutions of one or more nucleotides relative to areference sequence.

As used herein, the terms “exogenous” or “heterologous” mean, withrespect to two or more genetic sequences, that the genetic sequences donot occur in the same physical relation to each other in nature and/ordo not naturally occur within the same genome. For example, a geneticconstruct can include a coding region which is operably joined to one ormore regulatory elements, and these sequences are consideredheterologous to each other if they are not operably joined in natureand/or they are not found in the same genome in nature. Similarly, agenetic construct which is introduced into a cell is consideredheterologous to that cell to the extent that it contains geneticsequences not found in that cell. In addition, a synthetically-producedgenetic sequence based upon a naturally occurring sequence, will beheterologous to the naturally-occurring sequence to the extent codonshave been altered and the synthetic sequence does not exist in nature.Allelic variants of a sequence in a species are not consideredheterologous to each other.

As used herein, the term “operably joined” refers to a covalent andfunctional linkage of genetic regulatory elements and a genetic codingregion which can cause the coding region to be transcribed into mRNA byan RNA polymerase which can bind to one or more of the regulatoryelements. Thus, a regulatory region, including regulatory elements, isoperably joined to a coding region when RNA polymerase is capable underpermissive conditions of binding to a promoter within the regulatoryregion and causing transcription of the coding region into mRNA. In thiscontext, permissive conditions would include standard intracellularconditions for constitutive promoters, standard conditions and theabsence of a repressor or the presence of an inducer forrepressible/inducible promoters, and appropriate in vitro conditions, asknown in the art, for in vitro transcription systems.

As used herein, the term “expression” refers to the process by which acoding sequence of a gene is transcribed into a primary mRNA transcript,the primary mRNA transcript is processed into a mature mRNA, and themature mRNA is translated into a protein. Expression can optionallyinclude post-translation modifications of the resulting polypeptide.

As used herein, the phrase “genetic construct encoding a CatSper4protein” means a recombinant DNA, RNA, or nucleic acid analog moleculewhich includes a genetic sequence encoding, or which is complementary toa genetic sequence encoding, the amino acid sequence of the CatSper4protein, and which is capable of being expressed in a cell which hasbeen transformed with the construct. The construct can express theCatSper4 protein transiently, or can stably integrate into the genome ofthe cell and express the protein conditionally or constitutively.

As used herein, the term “vector” means any genetic construct, such as aplasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.,which is capable transferring gene sequences between cells. Vectors canbe capable of one or more of replication, expression, and insertion orintegration, but need not possess each of these capabilities. Thus, theterm includes cloning, expression, homologous recombination, andknock-out vectors.

As used herein, with respect to genetic engineering, the term“transform” means to introduce into a cell or an organism an exogenousnucleic acid or nucleic acid analog which replicates within that cell ororganism, that encodes a polypeptide sequence which is expressed in thatcell or organism, and/or that is integrated into the genome of that cellor organism so as to affect the expression of a genetic locus. The term“transform” is used to embrace all of the various methods of introducingsuch nucleic acids or nucleic acid analogs, including, but not limitedto the methods referred to in the art as transformation, transfection,transduction, electroporation, ballistic injection, and the like.

As used herein, a “nucleic acid analog” means a molecule havingsufficient structural and functional similarity to a nucleic acid todirect sequence-specific forward or reverse transcription ofcomplementary nucleic acids, or to direct sequence-specific translationof an encoded polypeptide within a living cell.

As used herein, the term “reporter gene” means any genetic sequencewhich, when expressed, has a biochemical or phenotypic effect which isdetectable. Reporter genes are also known in the art as “marker” genes.

As used herein, the term “antibody” is intended to embrace naturallyproduced antibodies, recombinantly produced antibodies, and antibodyfragments such as Fab fragments, F(ab′)₂ fragments, Fv fragments, andsingle-chain Fv fragment (scFv).

As used herein, the term “effective amount” of an agonist or antagonist,or an enhancer or repressor, means the total amount of the activecomponent(s) of a composition that is sufficient to cause astatistically significant change on a detectable biochemical orphenotypic characteristic. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the effect, whetheradministered in combination, serially or simultaneously.

As used herein, the term “substantially pure” means a preparation whichcontains at least 60% (by dry weight) of the protein of interest,exclusive of the weight of other intentionally included compounds.Preferably the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by dry weight of the protein ofinterest, exclusive of the weight of other intentionally includedcompounds. Purity can be measured by any appropriate method, e.g.,column chromatography, gel electrophoresis, amino acid compositionalanalysis or HPLC analysis. If a preparation intentionally includes twoor more different proteins of the invention, a “substantially pure”preparation means a preparation in which the total dry weight of theprotein of the invention is at least 60% of the total dry weight,exclusive of the weight of other intentionally included compounds.Preferably, for such preparations containing two or more proteins of theinvention, the total weight of the proteins of the invention should beat least 75%, more preferably at least 90%, and most preferably at least99%, of the total dry weight of the preparation, exclusive of the weightof other intentionally included compounds. Thus, if the proteins of theinvention are mixed with one or more other compounds (e.g., diluents,detergents, excipients, salts, sugars, lipids) for purposes ofadministration, stability, storage, and the like, the weight of suchother compounds is ignored in the calculation of the purity of thepreparation.

As used herein, the term “contacted” as in the phrase “A is contactedwith B,” means that A and B are brought into sufficient physicalproximity to interact at the molecular level, as by mixing A and Btogether in a solution, or pouring a solution of A over B on substrate.As used herein, the phrase “A is contacted with B” is intended to beequivalent to “B is contacted with A” and is not intended to imply thateither element is fixed relative to the other, or that either element ismoved relative to the other.

As used herein, the terms “modulate” or “affect” mean to either increaseor decrease. As used herein, the terms “increase” and “decrease” mean,respectively, statistically significantly increase (i.e., p<0.1) andstatistically significantly decrease (i.e., p<0.1).

General Considerations.

The present invention depends, in part, upon the identification,isolation and characterization of a cation channel protein which isexpressed in sperm cells, but not in other tissues tested, and whichplays a significant role in the motility of sperm and their ability tofertilize ova. The protein has been designated CatSper4 to indicate thatit is the fourth Cation channel which is Sperm-specific to beidentified.

The CatSper4 gene encodes six transmembrane domains and a pore region.The gene product is exclusively expressed in the testis and not in othertissues such as the heart, brain, spleen, lung, liver, skeletal muscle,or kidney. In sperm, the channel is localized primarily to the tail'sprincipal piece, not the head or midpiece.

Agonists and antagonists of the activity of the CatSper4 protein canmodulate sperm motility and, therefore, can be used to treat infertilityor as male and female contraceptives. In particular, CatSper4 representsan excellent target for non-hormonal contraceptives for both males andfemales, including humans and other mammals. Identification of theCatSper4 gene and protein also presents new targets for the diagnosisand treatment of infertility, and thus provides for new assays for theidentification of compounds that can modulate fertility.

CatSper4 Nucleic Acids.

In one aspect, the present invention provides nucleic acid molecules, ornucleic acid analogs, encoding the CatSper4 proteins, or usefulfragments thereof. The full length cDNA of the human CatSper4 gene isdisclosed as SEQ ID NO: 1. The full-length cDNA sequence of a murinehomolog is disclosed as SEQ ID NO: 3. The 5′ regulatory region of ahuman allele, including 6358 bases extending 5′ from the translationalstart codon, is disclosed as SEQ ID NO: 5. The 5′ untranslated region ofthis human allele is disclosed as SEQ ID NO: 6. The 3′ untranslatedregion of this human allele, including 72 bases extending 3′ from thetranslational termination codon, is disclosed as SEQ ID NO: 7.

Nucleic acid molecules of the invention can be DNA or RNA molecules, orhybrid DNA-RNA molecules. The nucleic acid analogs of the invention canbe any of those known in the art, such as peptide nucleic acids, analogsincluding modified bases (e.g., 2′-halogeno-2′-dexynucleosides) and/oranalogs including modified internucleoside linkages (e.g.,phosphorothioate linkages), which are useful in applications such as invitro translation or antisense technologies. In the remainder of thisdisclosure and the appended claims, whenever the term “nucleic acids” isused, the term is intended to embrace nucleic acid analogs when suchanalogs would be useful or suitable in the context of the usage. Thenucleic acids can be sense molecules corresponding to all or a portionof a CatSper4 gene sequence, or can be antisense molecules which arecomplementary to all or a portion of a CatSper4 gene sequence. Thenucleic acids can be derived from or correspond to genomic DNA or cDNA,or can be synthetic molecules based upon a CatSper4 protein sequence andthe genetic code (e.g., synthetic nucleic acids which reflect the codonusage preferences in the host cells used in an expression systems).

In some embodiments, the CatSper4 nucleic acids comprise the entirecoding region of a CatSper4 gene (e.g., SEQ ID NO: 1 or SEQ ID NO: 3).Such nucleic acids can be used to produce genetic constructs fortransformation of cells, or for in vitro transcription and translationsystems. Such nucleic acids can also be used as probes in hybridizationassays to detect CatSper4 sequences in samples of other nucleic acids.

In other embodiments, subsets of the CatSper4 nucleic acid sequences areprovided for use as primers for nucleic acid amplification reactions, asprobes in hybridization assays to detect CatSper4 sequences in samplesof other nucleic acids, or as probes to distinguish normal or wild-typesequence from abnormal or mutant sequences. In these embodiments, thenucleic acids of the invention comprise at 10, preferably at least 12,more preferably at least 14 and most preferably at least 16 consecutivenucleotides selected from a CatSper4 sequence such as SEQ ID NO: 1.Depending upon the nature of the application, it may be preferable tochoose CatSper4 sequences which will have unique targets, or which areexpected to have unique targets, within a sample being probed oramplified. Thus, for example, sequences which are longer and sequenceswhich do not include frequently repeated elements (for example,polyadenylation signals) are more likely to be uniquely representedwithin any given sample. For purposes of choosing primers foramplification reactions, sequences of at least 15, and preferably 18-25nucleotides are preferred.

In certain preferred embodiments, nucleic acids are provided whichencode structural domains of a CatSper4 protein, or which encodefragments of the protein which can serve as epitopes for the generationof antibodies. Thus, for example, preferred nucleic acids include thoseencoding the transmembrane domains of the CatSper4 proteins (i.e.,approximately residues 46-75, 86-98, 108-137, 155-202, 209-233 and242-272 of SEQ ID NO: 2, approximately residues 48-73, 81-111, 113-134,156-204, 208-233 and 245-273 of SEQ ID NO: 4, and allelic variants andhomologs thereof), encoding the extracellular loops betweentransmembrane domains (i.e., approximately residues 76-85, 138-154 and234-242 of SEQ ID NO: 2, approximately residues 74-80, 135-155 and234-244 of SEQ ID NO: 4, and allelic variants and homologs thereof), orencoding the pore region (i.e., approximately residues 222-231 of SEQ IDNO: 2, approximately residues 222-231 of SEQ ID NO: 4, and allelicvariants and homologs thereof). Other preferred nucleic acid acidsinclude those encoding epitopes of the CatSper4 proteins having highpredicted antigenicity, as identified by standard sequence analysistechniques described below. Thus, for example, preferred nucleic acidsinclude those encoding sequences within a high predicted antigenicityepitope of SEQ ID NO: 2, or within a high predicted antigenicity epitopeof SEQ ID NO: 4, and allelic and mammalian homologs thereof.

In certain embodiments, nucleic acids are provided which encodepolypeptides have at least 80%, and preferably at least 85%, 90% or 95%amino acid sequence identity with at least a structural domain of aCatSper4 protein. Thus, in some embodiments, a nucleic acid is providedwhich encodes a polypeptide having at least 80%, 85%, 90% or 95% aminoacid sequence identity with a transmembrane domain of a CatSper4proteins (e.g., approximately residues 46-75, 86-98, 108-137, 155-202,209-233 and 242-272 of SEQ ID NO: 2, approximately residues 48-73,81-111, 113-134, 156-204, 208-233 and 245-273 of SEQ ID NO: 4, andallelic variants and homologs thereof), an extracellular loop betweentransmembrane domains (e.g., approximately residues 76-85, 138-154 and234-242 of SEQ ID NO: 2, approximately residues 74-80, 135-155 and234-244 of SEQ ID NO: 4, and allelic variants and homologs thereof), ora pore region (e.g., approximately residues 222-231 of SEQ ID NO: 2,approximately residues 222-231 of SEQ ID NO: 4, and allelic variants andhomologs thereof). In some preferred embodiments, nucleic acids areprovided encoding a polypeptide having at least 80%, 85%, 90% or 95%amino acid sequence identity with a CatSper4 protein and having CatSper4activity. The ability of a protein to exhibit CatSper4 activity can bemeasured by its ability to complement a CatSper4−/− mutant (e.g., aCatSper4 knock-out mutant) and restore a normal or CatSper4+/+ phenotype(e.g., to restore sperm motility) in a cell otherwise capable ofexpressing CatSper4 activity (e.g., a sperm cell from the CatSper4−/−mutant).

In other embodiments, isolated nucleic acids are provided which includea nucleotide sequence that hybridizes to at least a portion of aCatSper4 coding sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 3) understringent hybridization conditions. Such conditions includehybridizations employing a wash step of 1.0×SSC at 65° C., andequivalents thereof. More stringent conditions can include wash steps of0.5×SSC, 0.2×SSC, or even 0.1×SSC. Other equivalently stringentconditions are well known in the art. See, e.g., Ausubel et al., eds.(1989), Current Protocols in Molecular Biology, Vol. I, John Wiley &Sons, Inc., New York. In preferred embodiments, the nucleic acid encodesa polypeptide having CatSper4 activity.

In another aspect, the invention provides nucleic acids, either isolatedor existing within cells, in which a nucleotide sequence encoding apolypeptide having CatSper4 activity is operably joined to aheterologous regulatory region such that the CatSper4 sequence isexpressed. Thus, in certain embodiments, a heterologous regulatoryregion can be inserted into a chromosome such that it is operably joinedto an endogenous CatSper4 sequence. In some embodiments, the polypeptidehas at least 80%, 85%, 90% or 95% amino acid sequence identity with anamino acid sequence of SEQ ID NO: 2 or 4. In other embodiments, thenucleic acid encoding the polypeptide hybridizes to at least a portionof a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 3 under conditionsincluding a wash step of 1.0×SSC at 65° C., 0.5×SSC, 0.2×SSC, or0.1×SSC.

In certain embodiments, the nucleic acids of the invention encodepolypeptides including a CatSper4 sequence of at least 50 amino acidresidues in length, and preferably at least 100, 200 or 300 amino acidresidues in length. These polypeptides can include a CatSper4 sequencewhich includes at least one transmembrane domain, at least oneextracellular loop domain, at least a pore region, or combinationsthereof. In some preferred embodiments, the polypeptide has CatSper4activity. Such activity can include induction of ion current; mediationof cAMP-induced Ca²⁺ influx; restoration of sperm motility whenexpressed in CatSper4−/− sperm; and/or restoration of the ability topenetrate eggs when expressed in CatSper4−/− sperm.

In another aspect, the invention provides kits for detecting at least aportion of a CatSper4 nucleic acid (i.e., CatSper4 genomic DNA, mRNA,cDNA or amplification products thereof). The kits include an isolatednucleic acid of the invention as a probe and means for detecting theprobe. The means for detecting the probe can be a detectable label boundto the probe or a secondary nucleic acid probe for detecting the firstprobe (e.g., labeled secondary nucleic acid which specificallyhybridizes to the isolated nucleic acid.).

Genetic Constructs.

In another aspect, the present invention provides genetic constructscomprising sequences selected from CatSper4 genes. In certainembodiments, the CatSper4 gene sequences are selected from the codingregion of the CatSper4 gene, and in other embodiments, the CatSper4 genesequences can be chosen from the CatSper4 regulatory regions extendingapproximately 500-1,500 bases, or approximately 600-1,000 bases, 5′ ofthe start codon, and approximately 250-1,000 bases, or approximately500-750 bases, 3′ of the termination codon.

In one series of embodiments, CatSper4 coding sequences (e.g., theentire coding region, sequences encoding structural domains, sequencesencoding potential epitopes, or sequences encoding useful primers orprobes) are operably joined to an endogenous or exogenous regulatoryregion to form an expression construct. Useful regulatory regions forthese purposes include the endogenous CatSper4 regulatory region,constitutive promoter sequences (e.g., CMV, SV40, EF2), induciblepromoter sequences (e.g., lacZ, tet). Many useful vector systems are nowwidely available. For example, useful bacterial vectors include, but arenot limited to, pQE70, pQE60, pQE-9 (Qiagen, Valencia, Calif.),pBluescript II (Stratagene, La Jolla, Calif.), and pTRC99a, pKK223-3,pDR540 and pRIT2T (Pharmacia, Piscataway, N.J.), pTrc (Amann et al.(1988), Gene 69:301-315) and pET 11d (Studier et al. (1990), Methods inEnzymol. 185:60-89). Examples of vectors for expression in yeast includepYepSec1 (Baldari et al. (1987), EMBO J. 6:229-234), pMFa (Kurjan et al.(1982), Cell 30:933-943), pJRY88 (Schultz et al. (1987), Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). TheCatSper4 proteins can also be expressed in insect cells (e.g., Sf 9cells) using, for example, baculovirus expression vectors including, butnot limited to, pAc vectors (Smith et al. (1983), Mol. Cell Biol.3:2156-2165) and pVL vectors (Lucklow et al. (1989), Virology170:31-39). Examples of mammalian expression vectors include, but arenot limited to, pCDM8 (Seed (1987), Nature 329:840) and pMT2PC (Kaufmanet al. (1987), EMBO J. 6:187-195). Other useful eukaryotic vectorsinclude, but are not limited to, pXT1, pSGS (Stratagene, La Jolla,Calif.), and pSVK3, pBPV, pMSG, and PSVLSV40 (Pharmacia, Piscataway,N.J.). Thus, one of ordinary skill in the art can choose a vector systemappropriate to the host cell to be transformed.

In other embodiments, the vectors comprise defective or partial CatSper4sequences in a “knock-out” vector. Such vectors are well-known in theart and can be used to produce a transgenic organism in which anendogenous gene is “knocked-out” by recombination with a partiallyhomologous exogenous sequence which introduces a mutation within theendogenous sequence. Typically, the vector is directed at an endogenoustarget sequences which can be all or part of a gene of interest. Thevector includes 5′ and 3′ flanking sequences which are homologous to the5′ and 3′ ends of the target. Between the 5′ and 3′ flanking sequencesis the sequence including the mutation. The mutation can be atermination mutation, frame-shift mutation, large deletion, or even theintroduction of a new coding sequence which serves both to disrupt theendogenous gene and to act as a marker for successful homologousrecombination. Knock-out vectors are further discussed below.

In other embodiments, the CatSper4 coding sequences can be joined toregulatory regions and heterologous coding sequences to form a geneticconstruct or fusion vector which encodes a fusion protein. Fusionvectors and fusion proteins can be useful to increase the expression ofthe CatSper4 protein, to increase the solubility of the CatSper4protein, and aid in the purification of the CatSper4 protein (e.g., byacting as a ligand for affinity purification). A proteolytic cleavagesite can be introduced at the junction of the CatSper4 and non-CatSper4protein sequences so that the CatSper4 protein can easily be separatedfrom the fusion moiety. Typical fusion expression vectors include pGEX(Smith et al. (1988), Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

In another series of embodiments, vectors or genetic constructs areproduced in which the coding region from a reporter gene is operablyjoined to the regulatory region of a CatSper4 gene (e.g., SEQ ID NO: 5and, optionally, SEQ ID NO: 6 at the 5′ end and, optionally, SEQ ID NO:7 at the 3′ end). Such genetic constructs are useful in assays toidentify or characterize compounds that enhance or repress CatSper4 geneexpression by enhancing or repressing transcription of the CatSper4gene. A wide variety of suitable reporter genes are known to those ofskill in the art, and are commercially available. Examples include, butare not limited to, the lacZ and luciferase genes.

Useful CatSper4 regulatory elements include sequences having at least80% nucleotide identity to at least 100 consecutive nucleotides selectedfrom SEQ ID NO: 5, preferably at least 200 consecutive nucleotides, andmore preferably at least 300-500 consecutive nucleotides. Usefulregulatory elements will retain the ability to promote transcription ofa coding sequence operably joined to the element in a mammalian cell inwhich a CatSper4 gene is expressed. In particular, useful regulatoryelements will retain the ability to promote transcription in cells inwhich the CatSper4 gene from which the element was derived is expressed,or in which a homolog of that CatSper4 gene is expressed.

Transformed Cell Lines.

In another aspect, the present invention provides cell lines transformedwith the nucleic acid molecules of the invention. Such cell lines cansimply propagate these nucleic acids (e.g., when transformed withcloning vectors) or can express the polypeptides encoded by thesenucleic acids (e.g., when transformed with expression vectors). Suchtransformed cell lines can be used to produce the CatSper4 proteins andCatSper4 fragments of the invention, or can be used in assays to screenfor compounds that enhance, repress, agonize, or antagonize CatSper4expression or activity.

The transformed cells can be produced by introducing into a cell anexogenous nucleic acid or nucleic acid analog which replicates withinthat cell, that encodes a polypeptide sequence which is expressed inthat cell, and/or that is integrated into the genome of that cell so asto affect the expression of a genetic locus. The transformation can beachieved by any of the standard methods referred to in the art astransformation, transfection, transduction, electroporation, ballisticinjection, and the like. The method of transformation is chosen to besuitable to the type of cells being transformed and the nature of thegenetic construct being introduced into the cells.

Preferred cell lines for transformation include bacterial cells (e.g.,Escherichia coli), yeast cells (e.g., Saccharomyces cerevisiae), insectcells (e.g., Drosophila melanogaster Schneider cells), nematode cells(e.g., Caenorhabditis elegans), amphibian cells (e.g., Xenopus oocytes),rodent cells (e.g., Mus musculus (e.g., murine 3T3 fibroblasts), Rattusrattus, Chinese Hamster Ovary cells (e.g., CHO-K1)), and human cells(e.g., human skin fibroblasts, human embryonic kidney cells (e.g.,HEK-293 cells), COS cells, spermatogonial cells). Transformed mammaliancells useful in the invention include somatic cells, fetal cells,embryonic stem cells, zygotes, gametes, germ line cells and transgenicanimal cells.

Appropriate cells can be transformed with any of the above-describedgenetic constructs in order to produce CatSper4 proteins, includingfragments of CatSper4 proteins, fusion proteins of CatSper4 proteins, ormarker proteins under the control of a CatSper4 regulatory region.

The cells can be transformed according to any method known in the artappropriate to the cell type being transformed. Appropriate methods caninclude those described generally in, e.g., Sambrook et al. (1989),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,New York; and Davis et al. (1986), Basic Methods in Molecular Biology,Elsevier. Particular methods include calcium phosphate co-precipitation(Graham et al. (1973), Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi (1980), Cell 22:479-488), electroporation(Shigekawa et al. (1988), BioTechniques 6:742-751), liposome-mediatedgene transfer (Mannino et al. (1988), BioTechniques 6:682-690),lipid-mediated transduction (Feigner et al. (1987), Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987), Nature 327:70-73).

Transgenic Animals.

The present invention also provides for the production of transgenicnon-human animal models in which wild type, allelic variant, chimeric,or antisense CatSper4 sequences are expressed, or in which CatSper4sequences have been inactivated or deleted (e.g., “knock-out”constructs) or replaced with reporter or marker genes (e.g., “knock-inreporter” constructs). The CatSper4 sequences can be conspecific to thetransgenic animal (e.g., murine sequences in a transgenic mouse) ortranspecific to the transgenic animal (e.g. human sequence in atransgenic mouse). In such a transgenic animal, the transgenic sequencescan be expressed inducibly, constitutively or ectopically. Expressioncan be tissue-specific or organism-wide. Engineered expression ofCatSper4 sequences in tissues and cells not normally containing CatSper4gene products can cause novel alterations of cation flux and lead tonovel cell or tissue phenotypes. Ectopic or altered levels of expressionof CatSper4 sequences can alter cell, tissue and/or developmentalphenotypes. Transgenic animals are useful as models of disorders arisingfrom defects in CatSper4 activity.

Transgenic animals are also useful for screening compounds for theireffects on CatSper4 activity. Transgenic animals transformed withreporter constructs can be used to measure the transcriptional effectsof small molecules or drugs or physical perturbations on the expressionof CatSper4 genes and proteins in vivo. The transgenic animals of theinvention, can be used to screen such compounds for therapeutic utility.

Animal species suitable for use in the animal models of the presentinvention include, but are not limited to, rats, mice, hamsters, guineapigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates(e.g., Rhesus monkeys, chimpanzees). For initial studies, transgenicrodents (e.g., mice) are preferred due to their relative ease ofmaintenance and shorter life spans. Transgenic non-human primates may bepreferred for longer term studies due to their greater similarity tohumans.

Using the nucleic acids disclosed and otherwise enabled herein, thereare several available approaches for the creation of a transgenicanimal. Thus, the enabled animal models include: (1) animals in whichsequences encoding at least a functional fragment of a CatSper4 gene hasbeen recombinantly introduced into the genome of the animal as anadditional gene, under the regulation of either an exogenous or anendogenous promoter element, and as either a minigene (i.e., a geneticconstruct of the CatSper4 gene based on cDNA with introns removed) or alarge genomic fragment; (2) animals in which sequences encoding at leasta functional fragment of a CatSper4 gene have been recombinantlysubstituted for one or both copies of the animal's endogenous CatSper4gene by homologous recombination or gene targeting; (3) animals in whichone or both copies of one of the animal's homologous CatSper4 genes havebeen recombinantly “humanized” by the partial substitution of sequencesencoding the human homolog by homologous recombination or genetargeting; (4) animals in which sequences encoding a reporter gene havereplaced the endogenous CatSper4 gene by homologous recombination; (5)and “knock-out” animals in which one or both copies of the animal'sCatSper4 sequences have been partially or completely inactivated by theinsertion, deletion or substitution of one or more nucleotides byhomologous recombination. These and other transgenic animals of theinvention are useful as models of infertility or other disorders arisingfrom defects in the CatSper4 gene and/or protein. These animals are alsouseful for screening compounds for their effects on the CatSper4 geneand/or protein.

To produce an animal model (e.g., a transgenic mouse), a wild type orallelic variant CatSper4 sequence or a wild type or allelic variant of arecombinant nucleic acid encoding at least a functional fragment of aCatSper4 protein is preferably inserted into a germ line or stem cellusing standard techniques of oocyte or embryonic stem cellmicroinjection, or other form of transformation of such cells.Alternatively, other cells from an adult organism can be employed.Animals produced by these or similar processes are referred to astransgenic. Similarly, if it is desired to inactivate or replace anendogenous CatSper4 sequence, homologous recombination using oocytes,embryonic stem or other cells can be employed. Animals produced by theseor similar processes are referred to as “knock-out” (inactivation) or“knock-in” (replacement) models.

For oocyte injection, one or more copies of the recombinant DNAconstructs of the present invention can be inserted into the pronucleusof a just-fertilized oocyte. This oocyte is then reimplanted into apseudo-pregnant foster mother. The live born animals are screened forintegrants using standard DNA/mRNA analysis (e.g., from the tail veinsof offspring mice) for the presence of the inserted recombinanttransgene sequences. The transgene can be either a complete genomicsequence introduced into a host as part of a yeast artificial chromosome(YAC), bacterial artificial chromosome (BAC), or other chromosome DNAfragment; as a cDNA with either the endogenous promoter or aheterologous promoter; or as a minigene containing all of the codingregions and other elements found to be necessary for optimum expression.

To create a transgene, the target sequence of interest (e.g., a wildtype or allelic variant of a CatSper4 sequence) is typically ligatedinto a cloning site located downstream of a promoter element which willregulate the expression of RNA from the sequence. Downstream of thecoding sequence, there is typically a polyadenylation sequence. Analternative approach to creating a transgene is to use an exogenouspromoter and regulatory sequences to drive expression of the transgene.Finally, it is possible to create transgenes using large genomic DNAfragments such as YACs which contain the entire desired gene as well asits appropriate regulatory sequences.

Animal models can be created by targeting endogenous CatSper4 sequencesfor homologous recombination. These targeting events can have the effectof removing endogenous sequence (knock-out) or altering the endogenoussequence to create an amino acid change associated with human disease oran otherwise abnormal sequence (e.g., a sequence which is more like thehuman sequence than the original animal sequence) (knock-in animalmodels). A large number of vectors are available to accomplish this andappropriate sources of genomic DNA for mouse and other animal genomes tobe targeted are commercially available (e.g., GenomeSystems Inc., St.Louis, Mo.).

The typical feature of these targeting vector constructs is that 2 to 4kb of genomic DNA is ligated 5′ to a selectable marker (e.g., abacterial neomycin resistance gene under its own promoter element termeda “neomycin cassette”). A second DNA fragment from the gene of interestis then ligated downstream of the neomycin cassette but upstream of asecond selectable marker (e.g., thymidine kinase). The DNA fragments arechosen such that mutant sequences can be introduced into the germ lineof the targeted animal by homologous replacement of the endogenoussequences by either one of the sequences included in the vector.Alternatively, the sequences can be chosen to cause deletion ofsequences that would normally reside between the left and right arms ofthe vector surrounding the neomycin cassette. The former is known as aknock-in, the latter is known as a knock-out.

Retroviral infection of early embryos can also be done to insert therecombinant DNA constructs of the invention. In this method, thetransgene (e.g., a wild type or allelic variant of a CatSper4 sequence)is inserted into a retroviral vector which is used to directly infectembryos (e.g., mouse or non-human primate embryos) during the earlystages of development to generate partially transgenic animals, some ofwhich bear the transgenes in germ line cells.

Alternatively, homologous recombination using a population of stem cellsallows for the screening of the population for successful transformants.Once identified, these can be injected into blastocysts, and aproportion of the resulting animals will show germ line transmission ofthe transgene.

Techniques of generating transgenic animals, as well as techniques forhomologous recombination or gene targeting, are now widely accepted andpracticed. A laboratory manual on the manipulation of the mouse embryo,for example, is available which details standard laboratory techniquesfor the production of transgenic mice (Hogan, et al. (1986),Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

CatSper4 Proteins and Polypeptides.

In another aspect, the present invention provides substantially purepreparations of CatSper4 proteins. The proteins can be isolated fromsperm cells, using standard techniques such as immunoaffinitypurification with the antibodies of the invention (see below), but arepreferably isolated from the transformed cells of the invention, inwhich they can be expressed at higher levels and, optionally, as fusionproteins which are more easily isolated and/or purified.

In some embodiments, the CatSper4 proteins comprise the entiretranslated sequence of the CatSper4 coding region. Examples of suchfull-length CatSper4 proteins include the human CatSper4 proteindisclosed as SEQ ID NO: 2 and the murine homolog disclosed as SEQ ID NO:4, as well as other CatSper4 proteins, including allelic and mammalianhomologs of these human CatSper4 proteins, and functional equivalentsthereof.

In other embodiments, the CatSper4 proteins are CatSper4 fragments. Suchfragments include the structural domains of the CatSper4 proteins,including the transmembrane, loop and pore-forming regions of theproteins. Preferred structural domains include the transmembrane domainsof the human CatSper4 protein (i.e., approximately residues 46-75,86-98, 108-137, 155-202, 209-233 and 242-272 of SEQ ID NO: 2,approximately residues 48-73, 81-111, 113-134, 156-204, 208-233 and245-273 of SEQ ID NO: 4, the extracellular loops between transmembranedomains (i.e., approximately residues 76-85, 138-154 and 234-242 of SEQID NO: 2, approximately residues 74-80, 135-155 and 234-244 of SEQ IDNO: 4), and the pore region (i.e., approximately residues 222-231 of SEQID NO: 2, approximately residues 222-231 of SEQ ID NO: 4), as well asallelic variants and homologs thereof. Other CatSper4 fragments includepotentially useful epitopes of the CatSper4 proteins, as identified bystandard sequence analysis techniques described below. Thus, forexample, preferred CatSper4 fragments include human CatSper4 sequenceswithin a high predicted antigenicity epitope of SEQ ID NO: 2, or withina high predicted antigenicity epitope of SEQ ID NO: 4, and allelic andmammalian homologs thereof.

In certain embodiments, polypeptides are provided having at least 80%,and preferably at least 85%, 90% or 95% amino acid sequence identitywith at least a structural domain of a CatSper4 protein. Thus, in someembodiments, a polypeptide is provided having at least 80%, 85%, 90% or95% amino acid sequence identity with a transmembrane domain of aCatSper4 proteins (e.g., approximately residues 46-75, 86-98, 108-137,155-202, 209-233 and 242-272 of SEQ ID NO: 2, approximately residues48-73, 81-111, 113-134, 156-204, 208-233 and 245-273 of SEQ ID NO: 4,and allelic variants and homologs thereof), an extracellular loopbetween transmembrane domains (e.g., approximately residues 76-85,138-154 and 234-242 of SEQ ID NO: 2, approximately residues 74-80,135-155 and 234-244 of SEQ ID NO: 4, and allelic variants and homologsthereof), or a pore region (e.g., approximately residues 222-231 of SEQID NO: 2, approximately residues 222-231 of SEQ ID NO: 4, and allelicvariants and homologs thereof). In some preferred embodiments,polypeptides are provided having at least 80%, 85%, 90% or 95% aminoacid sequence identity with a CatSper4 protein and having CatSper4activity. The ability of a protein to exhibit CatSper4 activity can bemeasured by its ability to complement a CatSper4−/− mutant (e.g., aCatSper4 knock-out mutant) and restore a normal or CatSper4+/+ phenotype(e.g., to restore sperm motility) in a cell otherwise capable ofexpressing CatSper4 activity (e.g., a sperm cell from the CatSper4−/−mutant).

In certain embodiments, the polypeptides of the invention include aCatSper4 sequence of at least 50 amino acid residues in length, andpreferably at least 100, 200 or 300 amino acid residues in length. Thesepolypeptides can include a CatSper4 sequence which includes at least onetransmembrane domain, at least one extracellular loop domain, at least apore region, or combinations thereof. In some preferred embodiments, thepolypeptide has CatSper4 activity. Such activity can include inductionof ion current when expressed in a cell (e.g., an oocyte); mediation ofcAMP-induced Ca²⁺ influx; restoration of sperm motility when expressedin CatSper4−/− sperm; and/or restoration of the ability to penetrateeggs when expressed in CatSper4−/− sperm.

Antibodies Against CatSper4 Proteins and Polypeptides.

In another aspect, the present invention provides substantially purepreparations of antibodies against the CatSper4 proteins, and methods ofmaking such antibodies. In particular, the invention provides antibodiesraised against CatSper4 epitopes having high predicted antigenicity,which therefore will selectively bind to and, thereby, isolate oridentify wild type and/or variant forms of the CatSper4 proteins.

The antibodies can be raised against the full-length CatSper4 proteins,against fragments of the CatSper4 proteins, or using any CatSper4epitope which is characteristic of the proteins and which substantiallydistinguishes them from other proteins. In preferred embodiments, theepitope is a protein sequence of at least 6-12, preferably 10-20, morepreferably 15-30 consecutive amino acid residues of a CatSper4 protein.In particular embodiments, the antibodies are raised against CatSper4epitopes selected from sequences within a high predicted antigenicityepitope of SEQ ID NO: 2, or within a high predicted antigenicity epitopeof SEQ ID NO: 4. Other preferred epitopes include allelic and mammalianhomologs of these epitopes. Epitopes having a high predictedantigenicity were identified by prediction of hydrophobicity, surfaceprobability and antigenic index using standard programs, including GCGand MacVector (Genetics Computer Group, University of WisconsinBiotechnology Center, Madison, Wis.; Accelrys Inc., San Diego, Calif.).See also, Jameson and Wolf (1988), Comput. Appl. Biosci. 4:181-186.

CatSper4 immunogen preparations can be produced from crude extracts(e.g., microsomal fractions of cells expressing the proteins), fromproteins or peptides substantially purified from cells which naturallyor recombinantly express them or, for small immunogens, by chemicalpeptide synthesis. The CatSper4 immunogens can also be in the form of afusion protein in which the non CatSper4 region is chosen for itsadjuvant properties and/or the ability to facilitate purification.

The antibodies of the invention can be polyclonal or monoclonal, or canbe antibody fragments, including Fab fragments, F(ab′)₂ fragments, Fvfragments, and single chain Fv fragments (scFv). In addition, afteridentifying useful antibodies by the method of the invention,recombinant antibodies can be generated, including any of the antibodyfragments listed above, as well as chimeric and/or humanized antibodiesbased upon non-human antibodies to the CatSper4 proteins. In light ofthe present disclosure of CatSper4 proteins, as well as thecharacterization of other CatSper4 proteins enabled herein, one ofordinary skill in the art can produce the above-described antibodies byany of a variety of standard means. For an overview of antibodytechniques, see Antibody Engineering, 2nd Ed., Borrebaek, ed., OxfordUniversity Press, Oxford (1995).

As a general matter, monoclonal anti-CatSper4 antibodies can be producedby first injecting a mouse, rabbit, goat or other suitable animal with aCatSper4 immunogen in a suitable carrier or diluent. Carrier proteins oradjuvants can be utilized, and booster injections (e.g., bi- ortri-weekly over 8-10 weeks) can be employed as necessary. After allowingfor development of a humoral response, the animals are sacrificed andtheir spleens are removed and resuspended in an appropriate buffer(e.g., phosphate buffered saline). The spleen cells serve as a source oflymphocytes, some of which will produce antibodies of the appropriatespecificity. These cells are then fused with an immortalized cell line(e.g., a myeloma), and the products of the fusion are plated into tissueculture wells in the presence of a selective agent (e.g., HAT). Thewells are serially screened and replated, selecting cells making auseful antibody each time. Typically, several screening and replatingprocedures are carried out until the wells contain single clones whichare positive for antibody production. Monoclonal antibodies produced bysuch clones can be purified by standard methods such as affinitychromatography using Protein A Sepharose, by ion-exchangechromatography, or by variations and combinations of these techniques.

Antibodies of the invention can be used in a variety of applications.For example, antibodies can be used in a purification process (i.e.,immunoaffinity purification) for CatSper4 proteins, in assays to detectthe presence or level of CatSper4 protein in sperm (e.g., in adiagnostic test for a CatSper4-related disorder), or in assays tomeasure the presence or level of CatSper4 expression in transformedcells (e.g., in assays for regulators of CatSper4 expression, in Westernblotting to identify cells expressing CatSper4 proteins, or inimmunocytochemistry or immunofluorescence techniques to establish thecellular or extracellular location of CatSper4 proteins).

The antibodies of the invention can be bound or conjugated with othercompounds or materials for diagnostic and/or therapeutic uses. Forexample, they can be coupled to labels such as radionuclides,fluorescent compounds (e.g., rhodamine), or enzymes for imaging ortherapy. The labels can be bound to the antibodies covalently ornon-covalently.

In another aspect, the invention provides kits for detecting at least anepitope of a CatSper4 protein. The kits include an anti-CatSper4antibody and a means for detecting the antibody. The means for detectingthe antibody can be a detectable label bound to the antibody orsecondary antibodies for detecting the anti-CatSper4 antibodies (e.g., alabeled goat anti-rabbit-Ig antibody as a secondary antibody fordetecting a rabbit anti-CatSper4 antibody).

Assays for Modulators of CatSper4 Expression or Activity.

In another aspect, the present invention provides assays for modulatorsof CatSper4 expression or activity. The modulators can affect thetranscription, translation, post-translational processing, localization,or activity of the CatSper4 gene and/or protein.

Thus, in one series of embodiments, the transformed cells of theinvention are contacted with a candidate compound, and the effect of thecompound on the expression or activity of CatSper4 is determined. As ageneral matter, the assays require contacting a candidate compound witha cell expressing a CatSper4 protein and measuring an indicator ofCatSper4 activity in the cell. The indicator can be an indicator oftranscription (e.g., mRNA levels), translation (e.g., protein levels),post-translational processing (e.g., specific glycosylation),localization (e.g., immunohistochemistry), or activity (e.g., sodium orother monovalent ion flux; calcium or other divalent ion flux). Theindicator measurement is then compared to a reference level to determinewhether the candidate compound caused an increase or decrease in theindicator. The reference level can be extrinsic (e.g., a predeterminedbaseline level) or intrinsic (e.g., a measurement of the same cell priorto contact with the candidate compound). If an increase or decrease issignificant (based on a single reading or on multiple readings from oneor more cells), the candidate compound is identified as a potentialmodulator of CatSper4 activity. Assays for changes in CatSper4 activitycan include any of those used routinely in the art for other genes. Forexample, changes in the presence or levels of CatSper4 mRNA or proteincan be detected to identify enhancers or repressors of CatSper4expression. Alternatively, when using a reporter gene construct of theinvention, the biochemical or phenotypic change characteristic of thereporter can be used as an indication that the candidate compoundenhances or represses reporter gene expression. In other embodiments,changes in the activity of the CatSper4 protein can be detected bymeasuring, for example, the flux of cations mediated by the CatSper4protein, or by measuring whole cell or channel currents. Measurements ofion fluxes can be facilitated by the use of chromophores which changecolor depending upon the concentration of specific ions. The effects ofcandidate compounds on mature sperm cells can be tested to confirm orvalidate results obtained in the transformed cells of the invention.

Compounds which bind to CatSper4 are candidates for modulating CatSper4activity. Thus, in another series of embodiments, libraries of compoundscan be screened to identify candidates for modulating CatSper4 activityby contacting candidate compounds with a CatSper4 protein, or at least astructural domain of a CatSper4 protein, to identify compounds that bindto CatSper4. CatSper4 structural domains which can be used in thesemethods include those described above (i.e., transmembrane domains,extracellular loops, pore regions), but extracellular loops and poreregions are preferred. In such methods, the CatSper4 protein or CatSper4structural domain can be immobilized (e.g., on a column ormicroparticle) and a solution of the candidate compound can be contactedwith the CatSper4 moiety, or the candidate compound can be immobilized(e.g., on a column or microparticle) and a solution of the CatSper4moiety can be contacted with the candidate compound. Alternatively, insome embodiments, neither the candidate compound nor the CatSper4 moietyis immobilized but, rather, both are in solution and binding is detectedby, for example, aggregation of particles bearing the binding partners.Binding can be detected by methods well known in the art (e.g.,radioactive or fluorescent labeling of one component of the potentialbinding pair; plasmon-resonance detection of binding; turbidity changesin aggregation assays). Compounds which, under physiological conditions(e.g., within the testis or epididymis, or within the vagina, uterus orfallopian tubes), exhibit significant binding (e.g., K_(d)≦10 μM) to aCatSper4 protein, are potential modulators of CatSper4 activity.

Methods of Modulating Fertility.

The CatSper4 gene and protein are ideal targets for potentialcontraceptive drugs. The restricted localization of CatSper4 to maturesperm means that a specific blocker should not affect other tissues andthus side effects should be low or nonexistent. Finally, since thechannel represents a novel structure, it may be an excellent target fornew channel agonists or antagonists.

Thus, in another aspect, the present invention provides methods ofdecreasing fertility by decreasing the expression or activity of aCatSper4 gene or protein. Such decreases in expression or activity canbe achieved by means of a small molecule which represses expression of aCatSper4 gene, by means of an antisense molecule which inhibits thetranslation of a CatSper4 mRNA, by means of a small molecule thatinterferes with CatSper4 translation or post-translational processing,by means of a small molecule that interferes with CatSper4 localization,or by means of a molecule which blocks CatSper4 activity as an ionchannel. Antibodies, including antibody fragments such as Fab fragments,F(ab′)₂ fragments, Fv fragments, and single-chain Fv fragments (ScFv),also can be used to inhibit CatSper4 activity by binding toextracellular domains of the protein and thereby block its activity.

Because most repressors or antagonists of CatSper4 expression oractivity will be reversible or will affect only mature sperm, theeffects of such compounds on fertility will be reversible because themolecules will be cleared from the body over time and new sperm areconstantly being produced. Thus, repressors or antagonists of CatSper4expression or activity can be used as human contraceptives because theycan cause reversible infertility. Such contraceptives can be takenorally or parenterally (e.g., injection, transdermal patch, orbioerodable implant) by females if they achieve sufficientconcentrations in the vagina, uterus or fallopian tubes to effectivelyinhibit CatSper4 activity and thereby decrease sperm motility and theability of sperm to penetrate the ZP. Similarly, such contraceptives canbe taken orally or parenterally by males if they achieve sufficientconcentration in the testes or seminal fluids to effectively inhibitCatSper4 expression or activity, and thereby decrease sperm motility andthe ability of sperm to penetrate the ZP. Alternatively, such compoundscan be formulated into lubricants, moisturizers, foams or jellies foruse with prophylactics, cervical caps, or contraceptive vaginal sponges,foams or jellies.

In another series of embodiments, repressors or antagonists of CatSper4genes and proteins can be used as contraceptives to treat non-humanmammals. These embodiments are similar to those described above forhuman contraception. Such contraceptives can be used with respect todomesticated animals that are maintained as pets, with respect to othercommercially valuable domesticated animals (e.g., cows, sheep, horses),or with respect to animal nuisances (e.g., mice, rats, raccoons,gophers). In some embodiments, the contraceptives are orally availableand can be mixed into food sources for the animals. In otherembodiments, the contraceptives can be administered parenterally (e.g.,injection, transdermal patch, or bioerodable implant).

To the extent that the mammalian CatSper4 genes and proteins and thefish, amphibian and insect homologs of the CatSper4 genes and proteinsshare substantial sequence identity, repressors or antagonists ofmammalian CatSper4 genes and proteins can also be used in the control offish, amphibian and insect nuisances (e.g., mosquitoes). In addition,the non-mammalian homologs of the CatSper4 genes and proteins can beused to identify additional repressors and antagonists which are morespecific or effective for such homologs.

Methods of CatSper4 Genotyping and Diagnosing CatSper4-RelatedDisorders.

In another aspect, the present invention provides methods for genotypingsubjects with respect to the CatSper4 gene, and diagnosingCatSper4-related disorders such as infertility. Thus, for example, theCatSper4 nucleic acids (or a portion thereof) of a subject can be testedto ascertain whether that subject's CatSper4 genotype includes anymutations in the sequences relative to wild-type. Of particularsignificance would be mutations which introduce termination orframe-shift mutations that prevent the production of functional CatSper4proteins. Point mutations, however, can also be identified which causedecreased CatSper4 activity. Similarly, the antibodies of the presentinvention can be used to test the sperm of a subject to determine thepresence or level of CatSper4 proteins. Of particular note would be anabsence or significant decrease in the level of CatSper4 protein. Pointmutations, however, can also cause infertility and can be detected byantibodies which are specific for epitopes including or affected by themutant sequences. Determination of a subject's CatSper4 genotype can beused for genetic or reproductive counseling, or for diagnosinginfertility that results from a CatSper4 defect.

To determine a subject's CatSper4 genotype, or for diagnosing aCatSper4-related disorder, the nucleic acids of the invention can beused as primers in polymerase chain reaction (PCR) (e.g., anchor PCR orRACE PCR), or ligase chain reaction (LCR) amplifications of thesubject's DNA/mRNA. See, e.g., U.S. Pat. No. 4,683,195 and U.S. Pat. No.4,683,202; Landegran et al. (1988), Science 241:1077-1080; Nakazawa etal. (1994), Proc. Natl. Acad. Sci. USA 91:360-364; and Abravaya et al.(1995), Nucleic Acids Res. 23:675-682. Other useful methods foramplifying a subjects DNA/mRNA using the nucleic acids of the inventioninclude self-sustained sequence replication (e.g., Guatelli et al.(1990), Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification (e.g., Kwoh et al. (1989), Proc. Natl. Acad. Sci. USA86:1173-1177), and Q-Beta Replicase-based systems (e.g., Lizardi et al.(1988), Bio/Technology 6:1197. The presence, absence or size of theresulting amplification products (e.g., Saiki et al. (1986), Nature324:163; Saiki et al. (1989), Proc. Natl. Acad. Sci. USA 86:6230; Gibbset al. (1989), Nucleic Acids Res. 17:2437-2448; Prossner (1993), Tibtech11:238; Gasparini et al. (1992), Mol. Cell Probes 6:1; Barany (1991),Proc. Natl. Acad. Sci. USA 88:189), direct sequencing of theamplification products (e.g., Maxim and Gilbert (1977), Proc. Natl.Acad. Sci. USA 74:560; Sanger (1977), Proc. Natl. Acad. Sci. USA74:5463), and other standard analytic techniques can be employed todetermine CatSper4 genotypes. The amplified products can also be used inmany of the techniques described below.

The nucleic acids of the invention also can be used as probes inhybridization and/or conformation-based assays to identify complementaryor imperfectly complementary sequences in a subject.

For example, in some embodiments, mutations can be identified byselectively hybridizing sample nucleic acids to immobilized controlnucleic acids. The controls can be adsorbed to filters or columns, orcan be arranged in high density ordered arrays containing hundreds orthousands of oligonucleotides probes (see, e.g., Cronin et al. (1996),Human Mutation 7:244-255; Kozal et al. (1996), Nature Medicine2:753-759).

In other embodiments, enzymatic or chemical cleavage can be employed tocleave or restrict duplexes of sample and control sequences atmismatched bases (e.g., Myers et al. (1985), Science 230:1242). Forexample, RNA/DNA duplexes can be treated with RNAse and DNA/DNA hybridscan be treated with 51 nuclease to digest duplexes at mismatched bases,G/A mismatches are cleaved at the A by the E. coli mutY enzyme, G/Tmismatches are cleaved at the T by the human thymidine DNA glycosylase(see, e.g., Hsu et al. (1994), Carcinogenesis 15:1657-1662). Chemicalcleavage of mismatches can be employed using, for example,hydroxylamine, osmium tetroxide and/or piperidine. See generally, e.g.,Cotton et al. (1988), Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992), Methods Enzymol. 217:286-295; and U.S. Pat. No. 5,459,039.

In other embodiments, mutations can create or destroy specific sequenceswhich serve cleavage points for restriction enzymes or ribozymes. Thus,restriction fragment length polymorphism (RFLP) analysis can be employedin which (amplified) sample DNA is digested with at least onerestriction endonuclease, and the resulting fragment lengths areanalyzed and compared to controls to determine the presence or absenceof mutations which affect the pattern of restriction fragment lengths.Similarly, sequence-specific ribozymes can be used to identify mutationsthat create or destroy ribozyme cleavage sites. See, e.g., U.S. Pat. No.5,498,531.

In other embodiments, mutations can be detected by their effect on theelectrophoretic mobility of a sequence, either as a single-strandednucleic acid or as duplex. For example, single-strand conformationpolymorphism (SSCP) analysis (Orita et al. (1989), Proc. Natl. Acad.Sci. USA 86:2766; Cotton (1993), Mutat. Res. 285:125-144; Hayashi(1992), Genet. Anal. Tech. Appl. 9:73-79; and Keen et al. (1991), TrendsGenet. 7:5). denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985), Nature 313:495). and temperature gradient gelelectrophoresis (Rosenbaum and Reissner (1987), Biophys. Chem.265:12753) can be employed.

These and other methods of detecting mutations in the CatSper4 genes andproteins will be apparent to one of ordinary skill in the art based uponthe nucleic acid and protein sequences disclosed herein.

In Vitro Fertilization.

In another aspect, the present invention provides a method of in vitrofertilization of ova by sperm characterized by decreased CatSper4expression or activity. It has previously been shown thatCatSper1-deficient sperm appear to be normal in all respects except intheir motility and their ability to penetrate the ZP. Moreover,CatSper1-deficient sperm are capable of fertilizing ova from which theZP has been removed. It is expected that CatSper4-deficient sperm willhave similarly reduced motility and ability to fertilize ova with intactZPs. Thus, the present invention provides a method of in vitrofertilization for CatSper4-deficient males in which the sperms of suchmales are treated to overcome the CatSper4 deficiency or are contactedwith ova from which the ZP have been removed. Because other geneticdeficiencies can result in sperm which are incapable of penetrating theZP, this method can be extended to other males having geneticdeficiencies which affect ZP-penetration or for which in vitrofertilization previously has been unsuccessful using ova with intactZPs.

Methods of Treating CatSper4-Mediated Infertility.

In another aspect, the present invention provides methods of treatinginfertility in CatSper4-deficient males, in which an enhancer ofCatSper4 expression or an agonist of CatSper4 activity is administeredto the subject. In other embodiments, gene or protein therapy can beemployed to provide the CatSper4 gene or protein to sperm (or spermprogenitors) which are deficient in the CatSper4 gene or protein. Forgene therapy, a genetic construct encoding a CatSper4 protein can beemployed to cause expression of a CatSper4 protein in sperm or spermprogenitors which are deficient in the CatSper4 gene or protein.

In another aspect, infertility of a mating pair (e.g., a human couple)can result from antibodies generated by the female against antigenspresent on the sperm of the male. In some cases, the antibodies can bedirected against an epitope of a CatSper4 protein. Thus, the presentinvention also provides methods of diagnosing an anti-CatSper4antibody-mediated infertility caused by anti-CatSper4 antibodies presentin a female urogenital tract. The methods include obtaining a sample ofantibodies present in the female and contacting the antibodies withCatSper4 proteins or fragments of CatSper4 proteins. In someembodiments, the CatSper4 fragments are epitopes of the CatSper4proteins having high predicted antigenicity (e.g., a high predictedantigenicity epitope of SEQ ID NO: 2, or a high predicted antigenicityepitope of SEQ ID NO: 4, and allelic and mammalian homologs thereof). Inthese methods, either the female's antibodies or the CatSper4proteins/fragments optionally can be immobilized and either the female'santibodies or the CatSper4 proteins/fragments optionally can bedetectably labeled to facilitate detection of binding between theantibodies and the CatSper4 proteins/fragments.

In these cases, administering an excess of the CatSper4 protein, or atleast a fragment of the CatSper4 protein including the relevant epitope,can saturate the binding sites of the anti-CatSper4 antibodies presentin the female's urogenital tract and thereby inhibit or reduce theantibody-mediated infertility. Alternatively, an anti-idiotypic antibody(i.e., an antibody which specifically binds to the variable regions ofanother antibody with a defined specificity) can be employed. That is,an antibody which binds specifically to anti-CatSper4 antibodies can beemployed to inhibit the anti-CatSper4 antibodies present in the female'surogenital tract and thereby inhibit or reduce the antibody-mediatedinfertility. One of ordinary skill in the art can easily identify therelevant CatSper4 epitopes recognized by such female antibodies (e.g.,using the methods described above) and produce substantially purepreparations of the relevant epitope or anti-idiotypic antibodies bystandard means. Thus, the invention also provides methods for treatingan anti-CatSper4 antibody-mediated infertility caused by anti-CatSper4antibodies present in a female urogenital tract. The methods includeadministering into the urogenital tract of the female an amount of therelevant CatSper4 epitope (or whole CatSper4 protein) or an amount ananti-idiotypic antibody effective to inhibit the anti-CatSper4antibodies and thereby inhibit or reduce the antibody-mediatedinfertility.

Business Methods Relating to CatSper4.

In another aspect, the present invention provides a method of conductinga drug discovery business comprising: identifying, by the assays of theinvention, one or more agents which antagonize CatSper4 activity;determining if an agent identified in such an assay, or an analog ofsuch an agent, inhibits at least one of sperm motility or eggpenetrance; conducting therapeutic profiling of an agent identified asan antagonist for efficacy and toxicity in one or more animal models;and formulating a pharmaceutical preparation including one or moreantagonist agents identified as having an acceptable therapeuticprofile.

In one embodiment, the drug discovery business further includes the stepof establishing a system for distributing the pharmaceutical preparationfor sale, and can optionally include establishing a sales group formarketing the pharmaceutical preparation.

In another aspect, the present invention provides a method of conductinga drug discovery business comprising: identifying, by the subject assay,one or more agents which agonize CatSper4 activity; determining if anagent identified in such an assay, or an analog of such an agent,increases at least one of sperm motility or egg penetrance; conductingtherapeutic profiling of an agent identified as an agonist for efficacyand toxicity in one or more animal models; and formulating apharmaceutical preparation including one or more agents identified ashaving an acceptable therapeutic profile.

In certain embodiments, the drug discovery business further includes thestep of establishing a system for distributing the pharmaceuticalpreparation for sale, and can optionally include establishing a salesgroup for marketing the pharmaceutical preparation.

In certain embodiments, the assay to identify agents which agonizeCatSper4 activity is conducted using wild type CatSper4. In anotherembodiment, the assay to identify agents which agonize CatSper4 activityis conducted using a mutant CatSper4. By a mutant CatSper4 is meant toinclude a CatSper4 polypeptide containing one or more insertions,deletions, or substitutions in amino acid sequence, wherein saidinsertions, deletions, or substitutions change the activity of themutant CatSper4 in comparison to wild type CatSper4. Such a change inactivity includes, but is not limited to, a change in motility, eggpenetrance, cation transport. A change in activity would also include achange in the proper localization or expression of the CatSper4 proteinor mRNA.

In still another aspect, the invention provides a method of conducting areproductive medicine business comprising: examining a sperm sample froma male patient, wherein said patient is experiencing a fertilityproblem; determining if said sperm are characterized by at least one ofa decrease in motility or a decrease in egg penetrance; performing invitro analysis to determine the efficacy of a CatSper4 agonist inincreasing at least one of sperm motility or egg penetrance;establishing a treatment regimen comprising administering an amount of aCatSper4 agonist effective to increase at least one of sperm motility oregg penetrance in said male.

In certain embodiments, the method further includes a step wherein saidmale patient is monitored by a physician to evaluate improvement infertility. Such evaluation can include examination of sperm at regularintervals following the initiation of treatment to measure improvementsin one or more of sperm motility or egg penetrance. The frequency offollow-up evaluation by the treating physician will be determined by thephysician or a trained health care provider. Factors to consider are thepatient's schedule and comfort level, as well as the urgency with whicha male patient is attempting to father an offspring. Representativefollow-up appointments can be conducted weekly, semi-weekly, or monthly.In another embodiment, the method further includes the step of billingthe patient or the patient's insurance provider. We note that in caseswhere the patient's health insurance is paying for all or a portion ofsaid fertility treatments, the policies of said health insuranceprovider will likely influence the frequency of follow-up appointments.

In yet another aspect, the present invention provides a method ofconducting a contraceptive medicine business comprising: providing apharmaceutical preparation discovered through the methods of a drugdiscovery business, wherein said preparation inhibits the activity ofCatSper4; providing instructions to physicians and health care providersfor the administration of an amount of said pharmaceutical preparationeffective to inhibit the activity of CatSper4, wherein said effectiveamount is sufficient to prevent pregnancy.

In one embodiment, the method further includes the step of establishinga system for distributing the pharmaceutical preparation for sale, andcan optionally include establishing a sales group for marketing thepharmaceutical preparation.

CatSper4 encodes a cation channel. Numerous types of cation channelsplay critical roles in cellular processes including regulation ofcardiac function (e.g., calcium channels). Thus, a great limitation ofmethods which employ administration of agents which either increase ordecrease the activity of cation channels is that such methods are likelyto have substantial side-effects. These side-effects can includesignificant cardiac complications. However, as disclosed herein,CatSper4 is specifically expressed in sperm. Accordingly, agents whichincrease or decrease the activity of CatSper4 can be administered topatients without the side effects associated with either general cationchannel antagonists and agonists, or antagonists and agonists of cationchannels which are more widely expressed in the body.

Through a drug discovery business, one or more agents which canantagonize the activity of CatSper4 can be identified. By antagonize theactivity is meant to decrease, in whole or in part, the activity ofCatSper4. Such a decrease in activity can be measured by examining atleast one of sperm motility, egg penetrance, or cation transport. Theterms decrease and antagonize will be used interchangeably throughout.

In certain embodiments, the initially identified CatSper4 agonist orantagonist can be subjected to further lead optimization, e.g., tofurther refine the structure of a lead compound so that potency andactivity are maintained but balanced with important pharmacologicalcharacteristics including:

-   -   Solubility    -   Permeability    -   Bioavailability    -   Toxicity    -   Mutagenicity    -   Pharmacokinetics—absorption, distribution, metabolism,        elimination of the drug

Structural modifications are made to a lead compound to address issueswith the parameters listed above. These modifications however, must takeinto account possible effects on the molecule's potency and activity.For example, if the solubility of a lead compound is poor, changes canbe made to the molecule in an effort to improve solubility; thesemodifications, however, can negatively affect the molecule's potency andactivity. SAR data are then used to determine the effect of the changeupon potency and activity. Using an iterative process of structuralmodifications and SAR data, a balance is created between thesepharmacological parameters and the potency and activity of the compound.

Candidate antagonists, or combinations thereof, must them be tested forefficacy and toxicity in animal models. Such therapeutic profiling iscommonly employed in the pharmaceutical arts. Before testing anexperimental drug in humans, extensive therapeutic profiling (e.g.,preclinical testing) must be completed to establish initial parametersfor safety and efficacy. Preclinical testing establishes a mechanism ofaction for the drug, its bioavailability, absorption, distribution,metabolism, and elimination through studies performed in vitro (that is,in test tubes, beakers, petri dishes, etc.) and in animals. Animalstudies are used to assess whether the drug will provide the desiredresults. Varying doses of the experimental drug are administered to testthe drug's efficacy, identify harmful side-effects that may occur, andevaluate toxicity.

Briefly, one of skill in the art will recognize that the identificationof a candidate agent which antagonizes CatSper4 activity in a drug basedscreen is a first step in developing a pharmaceutical preparation usefulas a contraceptive agent. Administration of an amount of saidpharmaceutical preparation effective to successfully prevent pregnancy(i.e., to act as a useful contraceptive agent) must be both safe andeffective. Early stage drug trials, routinely used in the art, help toaddress concerns of the safety and efficacy of a potentialpharmaceutical. In the specific case of a CatSper4 antagonist, efficacyof the pharmaceutical preparation could be readily evaluated in a mouseor rat model. Briefly, male mice could be administered varying doses ofsaid pharmaceutical preparations over various time schedules. Controlmale mice can be administered a placebo (e.g., carrier or excipientalone). The male mice are then allowed to mate freely by placing saidmale into cages with female mice, and measuring rate of conception overtime. Given the efficacy of currently available forms of birth control,an effective contraception should be at least 80% effective, preferably85% effective, more preferably 90% effective, most preferably 95%, 96%,97%, 98%, 99% or greater than 99% effective in preventing pregnancy.

In one embodiment, the step of therapeutic profiling includes toxicitytesting of compounds in cell cultures and in animals; analysis ofpharmacokinetics and metabolism of the candidate drug; and determinationof efficacy in animal models of diseases. In certain instances, themethod can include analyzing structure-activity relationship andoptimizing lead structures based on efficacy, safety and pharmacokineticprofiles. The goal of such steps is the selection of drug candidates forpre-clinical studies to lead to filing of Investigational New Drug(“IND”) applications with the U.S. FDA and/or similar applications withsimilar regulatory authorities prior to human clinical trials.

Between lead optimization and therapeutic profiling, one goal of thesubject method is to develop a CatSper4 agonist or antagonist which hasminimal side-effects. In the case of antagonists, the lead compoundswill have clinically acceptable effects on vasodilatation (i.e.,dizziness, hypotension, headache, flushing, edema, etc.), myocardialischemia, hypotension, bradycardia, transient asystole, exacerbation ofheart failure, ventricular dysfunction, SA node or AV conductiondisturbances, or plasma digoxin levels.

By “toxicity profiling” is meant the evaluation of potentially harmfulside-effects which may occur when an effective amount of apharmaceutical preparation is administered. A side-effect may or may notbe harmful, and the determination of whether a side effect associatedwith a pharmaceutical preparation is an acceptable side effect is madeduring the regulatory approval process. This determination does notfollow hard and fast rules, and that which is considered an acceptableside effect varies due to factors including: (a) the severity of thecondition being treated, and (b) the availability of other treatmentsand the side-effects currently associated with these availabletreatments. For example, the term cancer encompasses a complex family ofdisease states related to mis-regulated cell growth, proliferation, anddifferentiation. Many forms of cancer are particularly devastatingdiseases which cause severe pain, loss of function of the effectedtissue, and death. Chemotherapeutic drugs are an important part of thestandard therapy for many forms of cancer. Although chemotherapeuticsthemselves can have serious side-effects including hair-loss, severenausea, weight-loss, and sterility, such side-effects are consideredacceptable given the severity of the disease they aim to treat.

In contrast, however, most currently available forms of birth control donot have significant side-effects. Thus, a pharmaceutical preparation ofa CatSper4 antagonist should have minimal toxicity and side-effects.Toxicity tests can be conducted in tandem with efficacy tests, and malemice administered effective doses of the pharmaceutical preparation canbe monitored for adverse reactions to the preparation. Potential adversereactions associated with a contraceptive agent may include loss of sexdrive and behavioral changes. Blood, urine, and fecal samples taken fromtreated mice can also be monitored to detect any potential adversechanges in immune, kidney, or liver function. Additionally, given thatCatSper4 is a cation channel, mice receiving said pharmaceuticalpreparation should also be monitored for any changes in cardiac functionindicative of cross reactivity of the CatSper4 antagonist with othercation channels.

Agents which antagonize CatSper4 activity, and which are proven safe andeffective in animal studies, can be formulated into a pharmaceuticalpreparation. Such pharmaceutical preparations can then be marketed,distributed, and sold as contraceptive agents.

Given the link between loss of CatSper4 activity and fertility, there issubstantial utility in agents which increase the activity of CatSper4 totreat male fertility problems. Many instances of infertility involveproblems linked to the male. Such male infertility issues include lowsperm count, poor sperm motility, and abnormal sperm morphology.Currently there are few effective treatments for male-associatedinfertility.

The first step in developing potentially successful treatments for maleinfertility is the identification of CatSper4 agonists. A CatSper4agonist is one or more agents which increase the activity of CatSper4.As explained above with respect to CatSper4 antagonists, agonists of theCatSper4 protein are also expected to have fewer potential side-effectsthan other cation channel agonists.

Methods for identifying agents which act as CatSper4 agonists areperformed largely as detailed for CatSper4 antagonists. However, apreferred CatSper4 agonist will increase one or more of sperm motilityor egg penetrance. Additionally, we note that when identifying aCatSper4 agonist, such an agent can agonize the activity of a wild typeCatSper4. In addition, or alternatively, such an agent can agonize theactivity of a mutant CatSper4. One or more agonists identified by thesemethods can then be tested for safety and efficacy, as outline in detailabove. Agents which are shown to be safe and effective in animal studiesare formulated into a pharmaceutical preparation.

We note that said CatSper4 agonists are not likely to be effective fortreating all male fertility problems. However, it is expected that someundetermined percentage of male fertility problems will be amenable totreatment using agonists of CatSper4 function. For example, a certainpercentage of male infertility which results in poor sperm motility islikely due to mutations in CatSper4. Given that CatSper4 is expressedspecifically in sperm, males possessing such a mutation would beexpected to have little or no additional medical problems, and thisexplains in part why infertility is often found in otherwise healthymen. Additionally, a CatSper4 agonist can improve sperm motilityoverall, and thus help compensate for poor sperm motility due to otherunrelated causes.

Conducting a Reproductive Medicine Business.

A pharmaceutical preparation including one or more agents which agonizethe activity of a wild type or mutant CatSper4 can be useful inestablishing a reproductive medicine business which provides treatmentfor candidate male patients experiencing fertility difficulties. Spermsamples provided by male patients are examined to determine ifinfertility in said male patients may be amenable to treatment with thepharmaceutical preparation. Patients whose sperm is characterized by adecrease in at least one of motility or egg penetrance may be eligiblefor treatment. Prior to treatment, sperm samples provided by the malepatients are tested in vitro with the pharmaceutical preparation tofurther assess whether said male is eligible for treatment. Thisadditional step of in vitro testing helps to alleviate unnecessarytreatment in males whose infertility is unlikely to be improved with theCatSper4 agonist.

Male patients whose sperm shows increased motility or egg penetrance invitro are eligible for fertility treatment including the pharmaceuticalpreparation including one or more CatSper4 agonist. The exact treatmentregimen will vary from patient to patient, and can be readily determinedby an experienced medical professional. However, the treatment regimenwill include administration of an amount of said pharmaceuticalpreparation effective to increase at least one of sperm motility or eggpenetrance in said treated male. In a preferred embodiment, the increasein sperm motility or egg penetrance will result in an increase infertility.

Pharmaceutical Preparations.

Pharmaceutically acceptable preparations comprising a therapeuticallyeffective amount of one or more of the identified agents (i.e.,antagonists or agonists) described above, formulated together with oneor more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention can be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds can besimply dissolved or suspended in sterile water.

The phrase “therapeutically effective amount” as used herein means thatamount of an agent or composition which is effective for producing somedesired therapeutic effect, at a reasonable benefit/risk ratioapplicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations. In certain embodiments, the pharmaceutical preparation isnon-pyrogenic, i.e., does not elevate the body temperature of a patient.

The term “pharmaceutically acceptable salts” in this respect, refers tothe relatively non-toxic, inorganic and organic acid addition salts ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds of theinvention, or by separately reacting a purified compound of theinvention in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977), J. Pharm. Sci. 66: 1-19.)

The pharmaceutically acceptable salts of the subject agents include theconventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the agents of the present invention can contain one ormore acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of theagents, or by separately reacting the purified agent in its free acidform with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al. (1977), supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations can conveniently bepresented in unit dosage form and can be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1% to about 99% of active ingredient, preferably from about 5% to about70%, most preferably from about 10% to about 30%.

Methods of preparing these formulations or compositions include the stepof bringing into association one or more agents of the present inventionwith the carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association one or more agents of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration can be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A agent of the presentinvention can also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions can also comprise buffering agents. Solid compositions of asimilar type can also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, can optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They can also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They can be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions can also optionally containopacifying agents and can be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms can contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, can contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration can be presented as a suppository,which can be prepared by mixing one or more agents of the invention withone or more suitable nonirritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the rectum or vaginal cavityand release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of one ormore agents of this invention include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive agents can be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which can be required.

The ointments, pastes, creams and gels can contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of an agent of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the subject compound inthe proper medium. Absorption enhancers can also be used to increase theflux of the subject agent across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which can be reconstituted into sterileinjectable solutions or dispersions just prior to use, which can containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which can beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form can be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described specificallyherein. Such equivalents are intended to be encompassed in the scope ofthe invention.

We claim:
 1. A transgenic non-human animal comprising a modificationinto the non-human animal, the modification comprising an insertion of anucleic acid encoding a CaSper4 protein, the CatSper4 protein comprisingat least 90% similarity to the amino acid sequence of SEQ ID NO: 2 orSEQ ID NO: 4, and wherein the CatSper4 protein has CatSper4 activity. 2.A transgenic non-human animal comprising a modification into thenon-human animal, wherein the modification is replacement of anendogenous CatSper4 protein sequence with a reporter or marker gene,wherein the CatSper4 protein comprises at least 90% similarity to theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO:
 4. 3. A transgenicnon-human animal comprising a modification into the non-human animal,the modification comprising inactivation of an endogenous CatSper4protein, wherein the CatSper4 protein comprises at least 90% similarityto the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:
 4. 4. Thetransgenic non-human animal of claim 2, wherein the modification is byhomologous recombination to result in expression of the reporter gene.5. The transgenic non-human animal of claim 2, wherein the modificationis by homologous recombination to result in expression of the markergene.
 6. The transgenic non-human animal of any one of claims 1-3,wherein the CatSper4 sequences are conspecific to the transgenic animal.7. The transgenic non-human animal of any one of claims 1-3, wherein theCatSper4 sequences are transpecific to the transgenic animal.
 8. Thetransgenic non-human animal of any one of claims 1-3, wherein the animalis a mouse, rat, hamster, guinea pig, rabbit, dog, cat, goat, sheep,pigs, or a non-human primate.
 9. The transgenic non-human animal of anyone of claims 1-3, wherein the animal is a mouse.
 10. The transgenicnon-human animal of any one of claims 1-3, wherein the animal is anon-human primate.